Dynamics of serum hepatitis C virus load and quasispecies complexity during antiviral therapy in patients with chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) infection is a dynamic process during which viral genetic variants continuously develop as a result of the virus adaptation to the host's immune system. The level of viremia and the complexity of the hypervariable region 1 (HVR 1) quasispecies of hepatitis C virus during antiviral therapy reflect the dynamic balance between the viral and host components in response to therapy. OBJECTIVE: The aim of the study was to evaluate the dynamics of HCV viremia and the complexity of the HVR 1 quasispecies during the induction phase of a triple combination therapy regimen in nonresponders to earlier anti-HCV treatment. STUDY DESIGN: Ten patients with chronic hepatitis C undergoing antiviral combination therapy with interferon-alpha, ribavirin, and amantadine were studied. The serum HCV RNA level was monitored by a quantitative RT-PCR assay up to 3 months after start of treatment. The HVR 1 quasispecies complexity was analysed by an "in house" nested RT-PCR mediated single-strand conformation polymorphism (SSCP) assay. RESULTS: Baseline serum HCV RNA levels ranged from 1.94x10(6) to 5.53x10(6) copies/ml. In all patients, HCV subtype 1b was found. At the start of therapy, the SSCP assay revealed a high complexity pattern (at least six SSCP bands) in all patients. None of the patients responded within 4 weeks of treatment, however, the serum HCV RNA level decreased by one to two logs in eight patients. At week 4 after start of treatment, there was a decrease of SSCP bands in five patients. In four patients, SSCP bands remained unchanged and in one patient SSCP bands increased. At month 3 after start of treatment, serum HCV RNA was not detectable in one patient. CONCLUSION: Because of the low number of patients involved in this study, prediction of therapeutical success based on the quasispecies complexity was not possible. Larger studies are urgently needed.

Prevention of venous thromboembolism with low doses of heparin in urologic patients.

The effectiveness of low doses of heparin was investigated for the prevention of clinically manifested thromboembolic disease. In the group of 125 patients, who received 5,000 units of subcutaneous heparin two hours before the operation, and postoperatively 15,000 units per day for seven to ten days, deep vein thrombosis was not observed, and in only 1 patient (0.8%) pulmonary embolism was suspected. Of 216 patients from the control group, pulmonary embolism was clinically diagnosed in 4 (1.9%), deep vein thrombosis in 7 (3.4%), and in 2 patients (0.9%) fatal pulmonary embolism was found at autopsy. The difference between total thromboembolic disease of the control group (6.2%) and heparinized group was highly significant (p less than 0.02). Postoperative bleeding was seen two times more often in the heparin group, but bleeding was not severe and was easily controlled.

Experience with bone marrow transplantation for severe aplastic anemia and leukemia. Two years experience with 31 patients in Zagreb (Yugoslavia).

Since 1983, twelve patients with severe aplastic anemia and nineteen patients with leukemia were treated with bone marrow transplantation. Six patients with severe aplastic anemia are still alive at 300 to 1,210 days after transplantation, and twelve patients with leukemia are alive at 199 to 671 days. The incidence of GvHD was relatively high (60%). Bacterial infections were the main causes of death.

Cyclosporin neurotoxicity in patients treated with allogeneic bone marrow transplantation.

Toxicity to the central nervous system was observed in 3 out of 15 patients receiving cyclosporin prophylaxis for graft-versus-host-disease after allogeneic bone marrow transplantation. Neurological clinical features included grand mal seizures, dysarthria and vestibular and cochlear toxicity. In all three patients, cyclosporin plasma levels were increased at the time of overt clinical neurological signs. The symptoms resolved quickly with the reduction of the cyclosporin dose.

Transfusion medicine in the new millennium.

Blood is a tissue like all other tissues, however, it has always had a special meaning for man. Blood used to be attributed characteristics of the man whose body it was circulating through. Attempts were made to influence the person's character, properties, or disease, such as strength, old age and illness, by the bloodletting or blood administration. Blood was for centuries considered an elixir of life, and was ascribed various meanings at different times, e.g., mystic, religious, symbolic, racial, patriotic, biological, medical, scientific, industrial, and economic. Nowadays, the meaning of blood is most pronounced in the science, medicine, and economy.

A perception of transfusion safety and risks.

Transfusion therapy is today safer than ever. However, administration of homologous transfusions still has a number of different, even serious potential hazards. Therefore, transfusion therapy should only be given when the reasons for it are clear, its benefits outweigh the hazards, and the patient has accepted them after being well informed about them.

Clinically significant red cell alloantibodies in patients with warm autoimmune hemolytic anemia.

It has been generally accepted that the prevalence of clinically significant red cell alloantibodies is higher in patients with warm autoimmune hemolytic anemia (AIHA) than in other patients. In the present study, immunohematologic testing was performed in 328 polytransfusion patients with internal diseases. The patients were divided into two groups, i.e., without and with clinical signs of warm AIHA. Identification of red cell antibodies was performed in nonadsorbed sera of all patients, and in autoadsorbed and auto- and alloabsorbed sera of patients with warm AIHA. In the AIHA group, antibodies indicating red cell specificity were detected in 38.5% and 24.3% of patients in nonadsorbed and autoadsorbed sera, respectively. Clinically significant red cell alloantibodies were demonstrated in 10.3% nonadsorbed sera of patients free from signs of warm AIHA and in 10.4% auto- and alloadsorbed sera of patients with signs of warm AIHA. Study results showed the prevalence of clinically significant red cell alloantibodies in patients with signs of warm AIHA depend on the method of identification used rather than on the enhanced immune response induced by the autoimmune process.

Evaluation of the enzyme test for the detection of clinically significant red blood cell antibodies during pregnancy.

In the Croatian transfusion medicine, no general agreement has yet been achieved whether red blood cell (RBC) Rhesus (Rh) antibodies detected during pregnancy only by enzyme tests can cause hemolytic disease of the newborn (HDN). Results of the detection of clinically significant RBC antibodies by low-ionic-strength additive solution antiglobulin test (LISS-IAT) and trypsin enzyme test in 22,947 pregnant women are presented. All pregnant women in whom clinically significant RBC antibodies (RBC-CSA) were detected by LISS-IAT and/or enzyme tests were followed and observed during pregnancy. The women who had enzyme-only anti-D antibodies in their serum were followed up during subsequent pregnancies. Out of 302 positive results obtained by both techniques, irregular clinically significant enzyme-only antibodies (anti-RhD and anti-RhE specificity) were detected in 14 (4.6%) pregnant women. None of 11 RhD positive newborns whose mothers had enzyme-only anti-D antibodies, had signs of HDN after delivery. In these 11 women, anti-D antibodies were detected by LISS-IAT in the first trimenon of subsequent pregnancy. Nine infants born from subsequent pregnancies to women who had previously had enzyme-only anti-D, had clinical signs of HDN. The authors concluded that there is no need for enzyme tests in prenatal testing because enzyme tests are not reliable in the prediction of HDN.

Alloimmunizations following blood transfusions.

A retrospective study was performed to estimate the rate of alloimmunization against red blood cell (RBC) antigens in hospitalized patients and the frequency and specificity of clinically significant alloantibodies (CSA) detected during routine pretransfusion testing of 10,641 blood samples. Clinically significant alloantibodies were found in 116 out of the 10,641 tested serums (1.09%). The incidence of CSA in two surgical groups of patients was 0.18% and 0.68%, respectively. In patients with hematologic diseases the frequency of CSA was 17.6%, in patients with uremic disease it was 14%, and in patients with cirrhosis hepatis it was 6.9%. The incidence of clinically significant alloantibodies in patients who had one to five exposures to RBC antigens was 5.1%; in patients who had six to fifteen exposures, 87.9%; and in patients with more than fifteen exposures, 6.8%. The most frequently found single alloantibodies were anti-Kell (22%), and the following irregular alloantibodies were found with decreasing frequency: anti-E (20%), anti-D (11%), anti-c (10%), anti-C (7%), anti-Kidd (6%). The most frequent alloantibodies found together with other alloantibodies had anti-Duffy specificity (80%), and the following irregular alloantibodies were found with decreased frequency: anti-C (70%), anti-c (57%), and anti-Kidd (50%).

Croatian blood transfusion service in prevention of HIV spread during the war.

Beside the war, an unfavorable epidemiological situation and a large number of foreign peace troops that entered the country without having been previously tested for infectious diseases, the number of AIDS cases in our country remained relatively low. The transfusion service played a considerable part in the prevention of HIV infection spread. Although the blood transfusion service was faced with higher demands for blood and blood products, throughout the period of the war not a single blood unit was imported and no single unit of blood components was transfused without having been previously tested for the presence of viral disease markers. Despite enormous economic difficulties, three new diagnostic tests were then introduced in our transfusion practice as a regular procedure: anti-HCV in 1993, anti-HIV 2 in 1994 and anti-HIV 1/0 in 1995.

Ochratoxin A in blood of healthy population in Zagreb.

Healthy blood donors from the city of Zagreb were checked for the presence of a nephrotoxic mycotoxin ochratoxin A (OTA) in the plasma. Samples of blood were collected in June, September, and December 1997, and March 1998, totalling 200 or 50 in each round. The concentrations of OTA were measured using high pressure liquid chromatography (HPLC) method (detection limit 0.2 ng OTA/ml of plasma). The frequency of OTA-positive samples (> 0.2 ng/ml of plasma) showed significant seasonal variation (P < 0.001). The frequency of OTA-positive samples was the highest in March (65%) and it gradually decreased towards December (12%). The high frequency of positive samples coincided with seasons favouring growth of moulds and production of toxins. The daily intake of OTA by healthy persons in Zagreb was estimated from the mean concentration of OTA in samples collected during the whole year (0.19 ng OTA/ml plasma). The estimated daily intake was 0.26 ng/kg b.w., that is, substantially below the tolerable daily intake proposed by World Health Organization (16.0 ng/kg b.w.).

[The effect of leukocytes and erythrocytes on the metabolism and function of thrombocytes during preservation of thrombocyte concentrate at 22 degrees centigrade]. / Utjecaj leukocita i eritrocita na metabolizam i funkciju trombocita tijekom konzerviranja trombocitnih koncentrata na 22 degrees C.

The effect of leukocytes and erythrocytes on metabolic and functional changes was studied in 70 platelet concentrates kept for 72 hours at 22 degrees C in plastic PVC bags. On separating the platelet-rich plasma, a portion of buffy coat (i.e. a layer of leukocytes and erythrocytes) was added to one of the two series of platelet concentrates. These platelet concentrates did not differ from platelet concentrates prepared by the usual method, either by the platelet volume or count (p less than 0.05). The mean leukocyte count was 2.42 x 10(9)/L and 21.2 x 10(9)/L in the control and study series, respectively. After a 72 hour storage, a statistically significant difference (p less than 0.05) in the increase of lactate and ammonium concentrations, and a decrease in the glucose concentration and plasma pH were observed in platelet concentrates with a higher leukocyte count. The metabolic changes were accompanied by a statistically significant fall in the hypotonic shock (p less than 0.05). The results obtained in this study indicate that leukocytes in platelet concentrates elicit an adverse effect upon the platelet metabolism and function in vitro. In order to maintain the pH value at 6.0 during a 72 hour storage, the leukocyte count in platelet concentrates should not exceed 15.6 x 10(9)/L.

[Auto-reactive anti-B antibodies and hemolysis in patients with type B blood after kidney transplantation from an O blood group donor]. / Autoreaktivna anti-B antitijela i hemoliza u bolesnika B krvne grupe nakon transplantacije bubrega od davatelja O krvne grupe.

The course and the treatment of the patient M.F., who after kidney transplantation, produced autoreactive antierythrocyte antibodies is described. The patient M.F. was B, Rh positive and the kidney donor was 0, Rh positive. On the 12th posttransplantation day severe hemolysis, anemia and bilirubinemia were observed. Beside anti-A, autoreactive anti-B antibodies were discovered in the B group patient and the direct antiglobulin test (DAT) was positive. The patient was treated with several transfusions of 0 blood group washed red cells, immunosuppressive drugs (Cyclosporin, Methylprednisolone) and with daily plasmaphereses. After the 5th plasmapheresis the serum became clear, autoreactive anti-B antibodies could not be detected in the serum, but DAT was still positive. At that time the function of the graft was adequate. Between 22nd and 30th posttransplantation day DAT became negative. Autoreactive, anti-B antibodies were generated by the donor's lymphocytes, that were transferred with the kidney graft and which continued to synthetize anti-A,B antibodies in the patients' circulation.

[The effect of Rh-D immunoprophylaxis with hyperimmune anti-D-immunoglobulin on the occurrence of RhD immunization in pregnancy]. / Utjecaj provodenja RhD-imunoprofilakse hiperimunim anti-D-imunoglobulinom na ucestalost RhD-imunizacija trudnica.

During prenatal immunohaematological examination in the period from January 1, 1991 to December 31, 1995, in the Croatian Institute of Transfusion Medicine we tested sera of 5107 RhD negative women. All of them had pregnancies in their medical history. The frequency of Rh immunization was 4.6% in 1991; 4.1% in 1992; 2.5% in 1993; 2.5% in 1994 and 2.4% in 1995. Rh immunization during the first pregnancy was observed in 0.46% of women, in 1.8% during the second, in 9.4% during the third, in 22.4% during the fourth pregnancy, and 33.8% in women with more than five pregnancies. In women that have no abortions in their medical history, anti-D alloantibodies were found with the frequency of 0.46% at the end of the first pregnancy, 1.2% at the end of the second pregnancy, 5.9% at the end of the third pregnancy, 14.3% at the end of the fourth pregnancy, and 15.3% in women with more than five pregnancies. The frequency of anti-D alloantibodies in women who in their medical history have only abortions is 3.4% after the first abortion, 10.5% after the second, 17.8% after the third and 20.8% after the fourth or more abortions. The frequency of antibodies of anti-D specificities in women who had abortions and births is 17.1% at the end of the third pregnancy, 26.2% at the end of the fourth pregnancy, and 42.7% after more than five pregnancies. The frequency of anti-D alloantibodies in women who were protected from Rh immunization by hyperimmune anti-D globulin is 1%. The obtained results demonstrate that prevention of Rh immunization by hyperimmune anti-D globulin does not comprise all the Rh negative women, and is especially inadequate after abortions and multiple pregnancies.

[Safety of transfusion therapy with special emphasis on inactivated viruses in products made from human blood]. / Sigurnost transfuzijskog lijecenja s posebnim osvrtom na inaktivaciju virusa u preparatima proizvedenim iz ljudske krvi.

The most common complication of blood transfusion therapy is the transfusion-transmitted infection. The decrease of the risk had been achieved by several measures such as: the change from paid to all voluntary donor system, education of the population and potential donor groups as regards the risk factors, and the selection of donors without the risk factors, testing of donor blood for the presence of the hepatitis B and C viruses, HIV and syphilis, fractionation of plasma with the partition of viruses and their inactivation. In spite of all these efforts zero risk blood transfusion therapy has not been achieved and it is very unlikely that it will be achieved in the future. Clinicians must have sufficient background information on different procedures for viral inactivation in order to be able to select the least risky blood product.

[Factor VIII:C activity during heat inactivation of viruses in cryoprecipitates]. / Ispitivanje aktivnosti faktora VIII:C tokom toplinske inaktivacije virusa u krioprecipitatu.

The transmission of HIV by Factor VIII concentrates is the cause of AIDS in hemophiliacs. The yield of Factor VIII in the concentrate depends on the purification level and the applied inactivation procedure of viruses. Because of the high concentration of fibrinogen and the other proteins in cryoprecipitate, heat inactivation of the lyophilized cryoprecipitate leads to a substantial loss of Factor VIII:C, a change of color and the loss of solubility. With the addition of the buffer solution, which stabilizes Factor VIII, lyophilized cryoprecipitate can be heat treated at 60 degrees C through 72 hours with a loss of 15% of the activity of Factor VIII. This procedure inactivates more than 10(5) of Sendai viruses in 24 hours and more than 10(6) of Newcastle viruses in 72 hours in 1 ml of cryoprecipitate. The yield of F VIII:C in cryoprecipitate is about 440 u/L of fresh frozen plasma, which is acceptable. Heat inactivated, lyophilized cryoprecipitate, therefore, should be the product of choice for the countries with a small number of donors and a shortage of fresh frozen plasma.

[Evaluation of the direct antiglobulin test]. / Evaluacija direktnog antiglobulinskog testa.

The validity of a direct antiglobulin test (DAT) was evaluated by testing 7645 blood samples from hospitalized patients. DAT was routinely done in 7201 blood samples sent for pretransfusion testing and 444 blood samples specifically sent for the examination of immune hemolysis. Positive DAT was discovered in 0.04% (3/7201) pretransfusion samples and in 3.83% (17/444) samples examined for immune hemolysis. In 16 of the samples with positive DAT, IgG antibodies with or without complements and in 4 samples only components of complements were detected on RBC. The cost of positive DAT in pretransfusion testing is 92 times higher than that of DAT during laboratory investigation of immune hemolysis. Due to a low frequency of positive DAT during pretransfusion testing, its cast and the fact that patients had no clinical signs of immune hemolysis, we advocate no use of a routine DAT during pretransfusion testing.

[The combined immunoenzyme test: anti-HIV-1 and -2 and anti-HTLV-1 in the detection of donors with retrovirus infections]. / Ispitivanje kombiniranog enzimskog imunoloskog testa: anti-HIV 1/2 i anti HTLV-1 u otkrivanju davatelja zarazenih retrovirusima.

Serums of whole blood donors, plasma donors, hemophiliacs, persons with risks behavior and normal population simultaneously were tested for markers of infectious diseases, anti-HIV-1 with Plivazim and anti-HIV-1/2 and anti-HTLV-1/2 with Roche Retrovirus EIA. The positive results were confirmed by immunofluorescence assay and Western blot. Nonspecific reactive serums were detected by Roche Retrovirus EIA and by Plivazim EIA, but there was no significant difference in the frequence of reactive results. Roche Retrovirus EIA test had specificity of 99.16% and sensibility of 97.56% as compared to Plivazim. Simultaneous testing of donors with a combined test for anti-HIV-1/2 and anti-HTLV-1 is equally reliable as testing with only anti-HIV-1.

[Pseudothrombocytopenia caused by ethylenediaminetetraacetic acid (EDTA) (case report)]. / Pseudotrombocitopenija uzrokovana etilendiamintetraoctenom kiselinom (EDTA) (prikaz bolesnice).

Pseudothrombocytopenia is a laboratory artefact that can introduce serious problems in diagnosis and treatment in patients with low platelet count. The most common reason for this artefact is in vitro platelet clumping in blood samples collected into ethilenediaminetetraacetic acid (EDTA) anticoagulant. The clumping activity is greater at temperatures less than 37 degrees C, and the EDTA concentrations required for clumping are 20 times below anticoagulant concentrations. In this article we described the case of a female patient with diagnosed EDTA induced pseudothrombocytopenia. The cause of incorrectly low platelet counts was proved by simultaneous analysis in blood samples collected into EDTA anticoagulant and into heparin as a control sample. Absences of incorrectly low platelet count in heparin sample and rapid decrease of platelet count in EDTA sample were noticed. Decrease in platelet count was accompanied by increase in the number of leukocytes, so called pseudoleukocytosis. Careful examination of blood film is necessary to establish correct diagnosis, promptly after the blood collection and approximately two hours later. It is important to verify formation of clumps two hours after the blood collection and also progressive reduction in the platelet count in EDTA sample. By blood assessment conducted in this concern it is possible to avoid severe misinterpretation in such patients.